NER: Biological Nanomachines: Assembly and Function of Protein-DNA Nanostructures at the Single-Molecule Level

通过进化，生物产生了非常多样化和高效的蛋白质集合。这些蛋白质组装成似乎无限种类的纳米生物结构，它们促进了广泛的化学过程；因此，它们构成了一个有趣的生物分子纳米机器集合。负责维持和操纵DNA的蛋白质是这一集合中的一个重要子集。它们与DNA结合并改变DNA结构的能力对所有生物功能都是必不可少的。这些蛋白质在包装DNA、高保真复制遗传信息、读取遗传密码并将其转换为RNA、产生遗传多样性以及保持基因组的遗传和结构完整性方面发挥作用。这个项目描述了一种新的实验方法来研究在这些生物过程中起作用的几个纳米机器的组装和功能。该方法包括使用一种新颖的多端口层流微流动细胞，通过荧光显微镜实时地直接显示这些纳米生物结构在单个光学捕获的DNA分子上的组装、分解和运动。该仪器将使人们能够容易地将单个光学捕获的DNA分子顺序地引入一系列反应条件，并使用多波长荧光显微镜实时可视化分子的结构/组装的变化。该仪器的成功开发将揭示有关DNA-蛋白质相互作用的结构和功能的重要信息，这些信息无法使用大型DNA分子集合获得(这些信息往往通过平均而丢失，或被竞争的分子间相互作用所掩盖)。这项研究将提供有关蛋白质如何发挥作用改变DNA结构的革命性新信息。此外，这些实验技术还可以应用于许多其他纳米级生物结构的研究。

Through evolution, biology has produced a remarkably diverse and efficient collection of proteins. These proteins assemble into a seemingly infinite variety of nanobiostructures, and they promote an extensive repertoire of chemical processes; hence, they comprise an interesting collection of biomolecular nanomachines. The proteins that are responsible for the maintenance and manipulation of DNA are one important subset of this collection. Their capacity to assemble with, and to alter the structure of, DNA is essential for all biological function. These proteins function in the packaging of DNA, the high-fidelity copying of genetic information, the reading of the genetic code and its conversion into RNA, the generation of genetic diversity, and the preservation of genetic and structural integrity of the genome. This project describes a new experimental approach to study the assembly and function of several of the nanomachines that function in these biological processes. The method involves the direct visualization by fluorescence microscopy, in real-time, of the assembly, disassembly, and movement of these nanobiostructures on single, optically-trapped DNA molecules using a novel, multi-port, laminar-flow, micro flow cell. This instrument will allows one to readily introduce an individual, optically-trapped DNA molecule sequentially into a series of reaction conditions, and to visualize the changes in structure/assembly of the molecules in real-time using multi-wavelength fluorescence microscopy. The successful development of this instrument will reveal important information about the structure and function of DNA-protein interactions that cannot be obtained using large ensembles of DNA molecules (where such information is often lost by averaging, or obscured by competing intermolecular interactions). This research will provide revolutionary new information about how proteins function to alter the structure of DNA. Furthermore, the experimental techniques can be applied to the study of many other nanoscale biostructures.